Toxoplasma gondii is a ubiquitous pathogen infecting an estimated one-third of the U.S. population and 10-90% of individuals worldwide (depending on the country, various sociological and behavioral factors, etc). This parasite replicates intracellularly in a wide range of cell types and can persist for years in latent (tissue) cyst form. In addition to Toxoplasma, the protozoan phylum Apicomplexa includes many other parasites of clinical and/or veterinary importance. Although the diseases caused by these organisms differ greatly in nature (compare malaria, for example, with toxoplasmosis, or coccidiosis), the pathogenic impact of all apicomplexan parasites is ultimately attributable to proliferation, which makes understanding parasite replication an important goal. All Apicomplexans replicate by a distinctive process in which multiple daughters assemble simultaneously within the mother cell (termed 'schizogony'). This application proposes to characterize in molecular terms the structure and composition of the cytoskeletal organelles that serve as the focal point for initiation of daughter assembly. We use Toxoplasma for these studies because (1) T. gondii normally forms only two parasites at a time, making studies on the morphology of replication much more tractable than in Plasmodium or Eimeria species, and (2) a wide range of cell biological and molecular genetic tools are now available for T. gondii. In particular, fluorescent protein reporters now permit virtually all known subcellular structures to be visualized in living parasites, and the efficiency of transient transfection permits rapid assessment of recombinant plasmid function (even for lethal transgenes). Imaging techniques permit the analysis of relationships between various subcellular organelles over time, using quantitative time-lapse video microscopy and image de-convolution, laser scanning confocal microscopy, fluorescence photo-bleaching and recovery, and laser ablation. Molecular genetic approaches permit the mutation of essentially any parasite gene by either random or targeted methods, and identification of the lesions responsible. We aim to determine what proteins are needed, the function of each protein, and how they are arranged in 3D to provide the scaffold for building a parasite.